As the site of protein biosynthesis, the ribosome is a central element in the process of gene expression, yet its action is only poorly understood because of its complexity. The purpose of this project is to elucidate the mechanism of action of the ribosome by examining the functional roles of individual ribosomal protein and RNA components and the relationships among various partial activities of the ribosome. The ultimate goal of these studies is to understand the mechanism of protein biosynthesis in cells of all types, including those of normal, malignant, and otherwise abnormal human tissues, and pathogenic microorganisms, though bacterial ribosomes are chosen as the subject for reasons of experimental expediency. Such information is of potential importance in the understanding and control of many pathological conditions in which gene expression is involved. By means of chemical modification of functional groups, in conjunction with techniques for the in vitro reconstitution of ribosomes from dissociated molecular components, minimal structural alterations will be introduced into the ribosome. The resulting functional lesions will be characterized in detail by means of a variety of partial reaction assays which measure aspects of ribosome activity. The altered molecular component, and in some cases the functional group, responsible for the defect will be identified by constructing ribsomes containing a single modified component. In this way functional roles will be assigned to idividual ribosomal proteins and RNA. In addition characterization of a variety of defective ribosomes produced in this way will elucidate the relationships among the partial activities. These experiments will concentrate on the large (50S) ribosomal subunit. These studies should provide not only a better understanding of protein synthesis, but some insight into the organization of complex subcellular structures in general.